Studies of enzyme mechanisms have indicated that histidine is one of the most important functional amino-acids in proteins. In solution the role of protein functional groups is conventionally but still usefully probed by chemical modification methods. Diethyl pyrocarbonate is probably the most useful and specific reagent for examining histidine function. Preliminary work in this laboratory has shown however that the reaction of diethyl pyrocarbonate with histidine residues, hitherto believed to yield, reversibly, a carbethoxyimidazole derivative, can, under certain conditions lead to an irreversible Bamberger cleavage of the imidazole ring. This finding can lead to ambiguity in the interpretation of the results of experiments using this reagent in protein modification. The object of the present proposed research is to investigate the conditions under which this cleavage reaction occurs in protein modifications and thus develop conditions whereby diethyl pyrocarbonate and a variety of analogs can be used specifically either for the reversible modification or the irreversible cleavage. The study of both of these reactions at histidine in known protein environments (in proteins where X-ray structures are available) will be used to examine in detail the factors affecting the chemical reactivity of histidine residues in proteins. The application of these reagents in protein sequencing, where specific protein cleavage adjacent to histidine residues is proposed, will also be explored as part of this work.